Fosfomycin is an old antibiotic that has a unique mechanism of action by inactivating bacterial UDP-N- acetylglucosamine enolpyruvyl transferase, needed for peptidoglycan chain formation of the cell wall. Fosfomycin is approved for the treatment of patients with uncomplicated urinary tract infection (UTI). It has maintained activity against Escherichia coli, the primary UTI pathogen across all ages, despite decades of use. Recent surveys suggest that over 95% of E. coli clinical isolates are susceptible to this agent. The rapid rise of resistance in E. coli to commonly used oral antimicrobial agents such as fluoroquinolones, trimethoprim- sulfamethoxazole and cephalosporins has compromised treatment options of UTI, leading to reevaluation of fosfomycin for its management. In 2011, an updated guideline for the treatment of acute uncomplicated UTI and pyelonephritis in women was published, recommending fosfomycin as one of the first-line agents for the treatment of uncomplicated UTI. The use of fosfomycin has since skyrocketed at our center, increasing from none in 2009 to nearly 400 hospitalized patients treated with this agent in 2013. The use of fosfomycin is empiric and assumes susceptibility of E. coli to this agent, since routine agar-based susceptibility testing is not feasible in most clinical laboratories. These circumstances have led to the common use of fosfomycin, yet virtually nothing is known about resistance to it. Mechanisms of resistance to fosfomycin include loss of functional transporters, increased production of UDP-N-acetylglucosamine enolpyruvyl transferase and production of fosfomycin-modifying enzymes, but no clinical data on resistance exist for the U.S. In this study, we propose to test the following hypotheses: 1) fosfomycin resistance in E. coli occurs primarily by production of FosA-type modifying enzymes or inactivation of fosfomycin transporters, and 2) fosfomycin-resistant E. coli population is generated in the gastrointestinal tract of the patients after exposure to this agent, primarily due to inactivation of transporters. To test these hypotheses, we propose to determine the mechanisms of fosfomycin resistance in E. coli clinical and experimental strains and the associated fitness costs, and to determine the rate of resistance development after exposure to fosfomycin in patients using clinical and surveillance E. coli isolates before and after treatment with fosfomycin. The proposed study builds upon our preliminary work that identified fosfomycin-resistant E. coli as an emerging antimicrobial-resistant pathogen of concern. Currently, clinically relevant data on fosfomycin resistance mechanisms and the risk of their propagation are lacking. We aim to leverage our strengths - track record on investigation of antimicrobial resistance mechanisms in E. coli and a large inpatient population treated with fosfomycin - to address these critical questions. The findings of this study will enable investigations of approaches to prevent and mitigate the impact of fosfomycin resistance before it is a widespread problem.